Altering the redox state of skeletal muscle by glutathione depletion increases the exercise-activation of PGC-1α

We investigated the relationship between markers of mitochondrial biogenesis, cell signaling, and antioxidant enzymes by depleting skeletal muscle glutathione with diethyl maleate (DEM) which resulted in a demonstrable increase in oxidative stress during exercise. Animals were divided into six groups: (1) sedentary control rats; (2) sedentary rats + DEM; (3) exercise control rats euthanized immediately after exercise; (4) exercise rats + DEM; (5) exercise control rats euthanized 4 h after exercise; and (6) exercise rats + DEM euthanized 4 h after exercise. Exercising animals ran on the treadmill at a 10% gradient at 20 m/min for the first 30 min. The speed was then increased every 10 min by 1.6 m/min until exhaustion. There was a reduction in total glutathione in the skeletal muscle of DEM treated animals compared to the control animals (P < 0.05). Within the control group, total glutathione was higher in the sedentary group compared to after exercise (P < 0.05). DEM treatment also significantly increased oxidative stress, as measured by increased plasma F2-isoprostanes (P < 0.05). Exercising animals given DEM showed a significantly greater increase in peroxisome proliferator activated receptor γ coactivator-1α (PGC-1α) mRNA compared to the control animals that were exercised (P < 0.05). This study provides novel evidence that by lowering the endogenous antioxidant glutathione in skeletal muscle and inducing oxidative stress through exercise, PGC-1α gene expression was augmented. These findings further highlight the important role of exercise induced oxidative stress in the regulation of mitochondrial biogenesis.

Enhancing permanganate chemiluminescence detection for the determination of glutathione and glutathione disulfide in biological matrices

Acidic potassium permanganate chemiluminescence enables direct post-column detection of glutathione, but its application to assess the redox state of a wider range of biological fluids and tissues is limited by its sensitivity. Herein we show that the simple on-line addition of an aqueous formaldehyde solution not only enhances the sensitivity of the procedure by two orders of magnitude, but also provides a remarkable improvement in the selectivity of the reagent towards thiols such as glutathione (compared to phenols and amino acids that do not possess a thiol group). This enhanced mode of detection was applied to the determination of glutathione and its corresponding disulfide species in homogenised striatum samples taken from both wild type mice and the R6/1 transgenic mouse model of Huntington's disease, at both 8 and 12 weeks of age. No significant difference was observed between the GSH/GSSG ratios of wild type mice and R6/1 mice at either age group, suggesting that the early disease progression had not significantly altered the intracellular redox environment.

Redox sulfur chemistry of the copper chaperone Atox1 is regulated by the enzyme glutaredoxin 1, the reduction potential of the glutathione couple GSSG/2GSH and the availability of Cu(I)

Glutaredoxins have been characterised as enzymes regulating the redox status of protein thiols via cofactors GSSG/GSH. However, such a function has not been demonstrated with physiologically relevant protein substrates in in vitro experiments. Their active sites frequently feature a Cys-xx-Cys motif that is predicted not to bind metal ions. Such motifs are also present in copper-transporting proteins such as Atox1, a human cytosolic copper metallo-chaperone. In this work, we present the first demonstration that: (i) human glutaredoxin 1 (hGrx1) efficiently catalyses interchange of the dithiol and disulfide forms of the Cys(12)-xx-Cys(15) fragment in Atox1 but does not act upon the isolated single residue Cys(41); (ii) the direction of catalysis is regulated by the GSSG/2GSH ratio and the availability of Cu(I); (iii) the active site Cys(23)-xx-Cys(26) in hGrx1 can bind Cu(I) tightly with femtomolar affinity (K(D) = 10(-15.5) M) and possesses a reduction potential of E(o)' = -118 mV at pH 7.0. In contrast, the Cys(12)-xx-Cys(15) motif in Atox1 has a higher affinity for Cu(I) (K(D) = 10(-17.4) M) and a more negative potential (E(o)' = -188 mV). These differences may be attributed primarily to the very low pKa of Cys23 in hGrx1 and allow rationalisation of conclusion (ii) above: hGrx1 may catalyse the oxidation of Atox1(dithiol) by GSSG, but not the complementary reduction of the oxidised Atox1(disulfide) by GSH unless Cu(aq)(+) is present at a concentration that allows binding of Cu(I) to reduced Atox1 but not to hGrx1. In fact, in the latter case, the catalytic preferences are reversed. Both Cys residues in the active site of hGrx1 are essential for the high affinity Cu(I) binding but the single Cys(23) residue only is required for the redox catalytic function. The molecular properties of both Atox1 and hGrx1 are consistent with a correlation between copper homeostasis and redox sulfur chemistry, as suggested by recent cell experiments. These proteins appear to have evolved the features necessary to fill multiple roles in redox regulation, Cu(I) buffering and Cu(I) transport.

Effects of omega-3 PUFA on the vitamin E and glutathione antioxidant defense system in individuals at ultra-high risk of psychosis

BACKGROUND: Oxidative stress and impaired antioxidant defenses are reported in schizophrenia and are associated with disturbed neurodevelopment, brain structural alterations, glutamatergic imbalance, increased negative symptoms, and cognitive impairment. There is evidence that oxidative stress predates the onset of acute psychotic illness. Here, we investigate the effects of omega-3 PUFA on the vitamin E and glutathione antioxidant defense system (AODS). METHOD: In 64 help-seeking UHR-individuals (13-25 years of age), vitamin E levels and glutathione were investigated before and after 12 weeks of treatment with either 1.2g/d omega-3 (PUFA-E) or saturated fatty acids (SFA-E), with each condition also containing 30.4mg/d alpha-tocopherol to ensure absorption without additional oxidative risk. RESULTS: In multivariate tests, the effects on the AODS (alpha-tocopherol, total glutathione) were not significantly different (p=0.13, p=0.11, respectively) between treatment conditions. According to univariate findings, only PUFA-E caused a significant alpha-tocopherol increase, while PUFA-E and SFA-E caused a significant gamma- and delta-tocopherol decrease. Total glutathione (GSHt) was decreased by PUFA-E supplementation. CONCLUSION: Effects of the PUFA-E condition on the vitamin E and glutathione AODS could be mechanisms underlying its clinical effectiveness. In terms of the vitamin E protection system, PUFA-E seems to directly support the antioxidative defense at membrane level. The effect of PUFA-E on GSHt is not yet fully understood, but could reflect antioxidative effects, resulting in decreased demand for glutathione. It is still necessary to further clarify which type of PUFA/antioxidant combination, and in which dose, is effective at each stage of psychotic illness.

The Glutathione System: a new drug target in neuroimmune disorders

Glutathione (GSH) has a crucial role in cellular signaling and antioxidant defenses either by reacting directly with reactive oxygen or nitrogen species or by acting as an essential cofactor for GSH S-transferases and glutathione peroxidases. GSH acting in concert with its dependent enzymes, known as the glutathione system, is responsible for the detoxification of reactive oxygen and nitrogen species (ROS/RNS) and electrophiles produced by xenobiotics. Adequate levels of GSH are essential for the optimal functioning of the immune system in general and T cell activation and differentiation in particular. GSH is a ubiquitous regulator of the cell cycle per se. GSH also has crucial functions in the brain as an antioxidant, neuromodulator, neurotransmitter, and enabler of neuron survival. Depletion of GSH leads to exacerbation of damage by oxidative and nitrosative stress; hypernitrosylation; increased levels of proinflammatory mediators and inflammatory potential; dysfunctions of intracellular signaling networks, e.g., p53, nuclear factor-κB, and Janus kinases; decreased cell proliferation and DNA synthesis; inactivation of complex I of the electron transport chain; activation of cytochrome c and the apoptotic machinery; blockade of the methionine cycle; and compromised epigenetic regulation of gene expression. As such, GSH depletion has marked consequences for the homeostatic control of the immune system, oxidative and nitrosative stress (O&NS) pathways, regulation of energy production, and mitochondrial survival as well. GSH depletion and concomitant increase in O&NS and mitochondrial dysfunctions play a role in the pathophysiology of diverse neuroimmune disorders, including depression, myalgic encephalomyelitis/chronic fatigue syndrome and Parkinson’s disease, suggesting that depleted GSH is an integral part of these diseases. Therapeutical interventions that aim to increase GSH concentrations in vivo include N-acetyl cysteine; Nrf-2 activation via hyperbaric oxygen therapy; dimethyl fumarate; phytochemicals, including curcumin, resveratrol, and cinnamon; and folate supplementation.